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Peculiar p53 mutated protein-p53N236S of ALT tumor caused by progeria syndrome and application thereof

A p53n236s and mutant protein technology, which is applied in the fields of application, antineoplastic drugs, peptide/protein components, etc., can solve the problem of little research on the function of p53N239S mutant protein

Inactive Publication Date: 2012-03-21
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are very few functional studies on the p53N239S mutant protein, most of which are functional predictions of numerous p53 mutation points in yeast (Robert, Michel et al.2000; Kato, Han et al.2003; Dearth, Qian et al.2007 )

Method used

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  • Peculiar p53 mutated protein-p53N236S of ALT tumor caused by progeria syndrome and application thereof
  • Peculiar p53 mutated protein-p53N236S of ALT tumor caused by progeria syndrome and application thereof
  • Peculiar p53 mutated protein-p53N236S of ALT tumor caused by progeria syndrome and application thereof

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Effect test

Embodiment 1

[0023] The p53A1243G mutation was only detected in cells that can give rise to ALT tumors, suggesting that this mutation is closely related to ALT tumorigenesis.

[0024] From progeria syndrome G5mTR - / - Wrn - / - The fibroblasts (mouse embryofibroblast, MEF) cultured from mouse embryos also showed the characteristics of the fibroblasts of WS patients, and the cells cultured in vitro quickly entered senescence. However, such senescent cells are prone to escape senescence and form immortalized cell clones, and some immortalized cell clones can form tumors in severe immunodeficiency mice (SCID mice) (Laud, Multani et al. 2005).

[0025] G5mTR - / - Wrn - / - Mice provide a genetically clear model for ALT tumor research. In order to study the molecular regulation mechanism of WS cells from senescence, escape from senescence to immortalization, and ALT tumorization, the present invention compared and studied three immortalized cells (3B-1, 3B- 2, 9B-1) and four immortalized cells (...

Embodiment 2

[0031] The p53A1243G mutation leads to the loss of the mutant protein p53N236S that inhibits the tumor growth function of wild-type p53, thus leading to tumorigenesis.

[0032] To establish a cell line stably expressing p53N236S for studying the functional changes of the mutant protein, p53- / -MEF cells were transfected with empty vector PQCXIP or a vector expressing p53N236S. After adding the antibiotic puromycin for 1-2 months, the cell clones stably expressing p53N236S were screened by Western blotting and immunofluorescence staining.

[0033] The above established cells expressing or not expressing the mutant protein p53N236S were treated with 10Gy ionizing radiation to extract cellular RNA, and then cDNA was synthesized with AMV first-strand cDNA synthesis kit (Invitrogen, CA). Real-time PCR to measure p21 before and after irradiation (Forward primer: 5'CCA GGC CAAGAT GGT GTC TT 3', Reverse primer: 5'TGA GAA AGG ATC AGC CAT TGC 3'), CyclinG (Forward primer: 5'CTTTGACACGGAG...

Embodiment 3

[0036] The mutant protein p53N236S acquires a new function and can cooperate with Ras to promote tumorigenesis.

[0037] It is worth noting that p53N236S does not just lose the function of p53, in p53 - / - The introduction of p53N236S into the cells made the cells highly sensitive to the induction of Ras oncogene and rapidly formed huge tumors. The specific method is as follows: p53- / -MEF cells were co-transfected with the empty vector PQCXIP or the vector expressing p53N236S, combined with the oncogene Ras expression vector. After 1-2 months of selection with the antibiotic puromycin, the cell clones stably expressing p53N236S and Ras proteins were screened by Western blotting. Expand the p53- / - MEF cell line stably expressing p53N236S and Ras protein ( Figure 5 p53- / -+S+Ras), or p53- / -MEF cell lines expressing Ras protein alone ( Figure 5 in p53- / -+vector+Ras), take 1x10 respectively 6 Cells / injection site, injected subcutaneously into 6 SCID mice (24 injection sites), ...

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Abstract

The invention relates to a mutant gene p53A1243G caused by the point mutation of a tumor suppressor gene p53, encoded protein p53N236S and functions thereof, and application thereof to tumor diagnosis. First, the important functions of p53N236S in an ALT tumor caused by progeria syndrome are disclosed. The invention can be applied to the tumor diagnosis with p53N236S as the target molecule, the screening of anticancer medicine and cancer treatment strategies, and has important significance in preventing tumors caused by aging.

Description

technical field [0001] Specifically, the present invention relates to a mutant protein p53N236S caused by a point mutation of the tumor suppressor gene p53 and its function. The discovery of the function of the mutant protein can be applied to tumor diagnosis, screening of antitumor drugs, and formulation of tumor treatment strategies. technology field. Background technique [0002] Human premature aging syndrome (Werner Syndrome, WS) is an autosomal recessive mutation genetic disease, manifested as obvious premature aging (premature aging) and shortened life span, the average survival age is only 46-48 years old. Common diseases also include soft tissue osteosarcoma (soft tissue sarcoma), premature atherosclerosis, hemorrhagic heart disease, osteoporosis, cataract, type 2 diabetes, etc. (Epstein, Martin et al. 1966). Salk et al. isolated fibroblasts from WS patients and found that the chromosomes of these cells were extremely unstable, presenting the so-called translocatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/47C12Q1/68C12Q1/02G01N33/53A61K49/00A61K48/00A61K38/17A61P35/00
Inventor 罗瑛
Owner KUNMING UNIV OF SCI & TECH
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